We have analyzed gene expression in mouse embryonic prostate stem cells and their niche, the urogenital sinus mesenchyme. In examining mRNAs for proteins that are expressed in prostate stem cells that have corresponding receptors or ligands expressed in UGM cells, we have found that ephrins and their receptors, the Eph proteins, are highly represented. As the ephrins and Eph receptors have recently been shown to regulate stem cells from other organs, we hypothesize that they may play an important role in signaling between adult prostate stem cells and their niche. We propose three specific aims to examine the role of ephrin and Eph receptors in prostate stem cell growth and differentiation. In the first specific aim, we will confirm by immunohistochemistry and FACS that the 4 ephrins and 8 Eph receptors differentially expressed in the embryonic niche are also expressed in the adult mouse prostate. We will also examine if purified ephrins or Eph receptors alter stem cell proliferation or differentiation in vitro. In the second specific aim, we will determine the effectof knocking down or increasing expression of one ephrin and one Eph receptor on the ability of stem cells to regenerate a prostate in vivo. In this assay, prostate stem cells will be co-implanted with modified or normal urogenital sinus mesenchyme cells under the renal capsule, where they are expected to form a prostate-like organ. Our choice of ephrin or Eph receptor to be knocked down or over expressed will be informed by the results of Aim 1. As prostate tumors are thought to arise from stem cell-like tumor-initiating cells, we will also examine the effect of knocking down or increasing ephrin or Eph receptor on the proliferation and tumor-forming ability of tumor-initiating cells in Aim 3. As a model of tumor-initiating cells, we will use mouse prostate Sca-1HI stem cells that have been transformed with activated AKT-1 as the AKT pathway is activated in more than 50% of human prostate tumors. These cells form prostate tissue with PIN lesions when inoculated under the renal capsule of recipient mice. We will determine the effect of decreased or increased expression of ephrins or Eph receptors on the tumor-forming ability of the transformed stem cells. These experiments will provide basic information about the role of ephrins and Eph receptors in signaling between stem cells or tumor-initiating cells and their niche. These studies may provide evidence that targeting ephrin-Eph signaling may disrupt prostate tumor-initiating cell signaling with the tumor niche, leading to new therapies for advanced prostate cancer.